Mutants of igf binding proteins and methods of production of antagonists thereof

ABSTRACT

The present invention provides a crystal suitable for X-ray diffraction, comprising a complex of insulin-like growth factor I or II (IGF) and a polypeptide consisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9 th  to 12 th  cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7 th  to 10 th  cysteine of IGFBP-6; methods for the determination of the atomic coordinates of such a crystal; IGFBP mutants with enhanced binding affinity for IGF-I and/or IGF-II, and methods to identify and optimize small molecules which displace IGFs from their binding proteins.

The present invention relates to a complex of an IGF binding proteinfragment (IGFBP) with IGF, its uses and to novel IGFBP mutants with ahigher affinity than natural IGFBPs for IGF as well as to methods forthe production of antagonists for IGFBPs which hinder or reverse complexformation between IGFBPs and IGF.

Introduction

Insulin-like growth factors I and II (hereafter also referred to as IGFsor IGF) are members of the insulin superfamily of hormones, growthfactors and neuropeptides whose biological actions are achieved throughbinding to cell surface receptors. IGF actions are regulated by IGFbinding proteins (IGFBPs) that act as transporters of IGFs, protect themfrom degradation, limit their binding to receptors, and maintain a“reservoir” of biologically inactive IGF (Martin, J. L., and Baxter, R.C., IGF binding proteins as modulators of IGF actions; in: Rosenfeld, R.G., and Roberts, C. T. (eds.), The IGF system, Molecular Biology,Physiology, and Clinical Applications (1999), Humana Press, Totowa, pp.227-255; Jones, J. L., and Clemmons, D. R., Endocr. Rev. 12 (1995)10-21; Khandwala, H. M., et al., Endocr. Rev. 21 (2000) 215-244; Hwa,V., et al., The IGF binding protein superfamily, In: Rosenfeld, R. G.,and Roberts, C. T. (eds.), The IGF system, Molecular Biology,Physiology, and Clinical Applications (1999), Humana Press, Totowa, pp.315-327). The IGF and growth hormone (GH) axis plays a large part inregulating fetal and childhood somatic growth and several decades ofbasic and clinical research have demonstrated that it also is criticalin maintaining neoplastic growth (Khandwala, H. M., et al., Endocr. Rev.21 (2000) 215-244). High circulating IGF-I concentrations may also be animportant determinant of cancer incidence (Hankinson, S. E., et al.,Lancet 351 (1998) 1393-1396; Holly, J., Lancet 351 (1998) 1373-1374;Wolk, A., Lancet 356 (2000) 1902-1903). Virtually every level of the IGFsystem mediating response on the tumor tissues (IGFs, IGFBPs, IGFreceptors) can be targeted for therapeutic approaches (Khandwala, H. M.,et al., Endocr. Rev. 21 (2000) 215-244; Fanayan, S., et al., J. Biol.Chem. 275 (2000) 39146-39151; Imai, Y., et al., J. Biol. Chem. 275(2000) 18188-18194). It should also be mentioned here that IGFBP-3 hasIGF-independent anti-proliferative and proapoptotic effects (Wetterau,L. A., et al., Mol. Gen. Metab. 68 (1999) 161-181; Butt, A. J., et al.,J. Biol. Chem. 275 (2000) 39174-39181).

IGF-I and IGF-II are 67% identical single polypeptide chains of 70 and67 amino acids, respectively, sharing with insulin about 40% sequenceidentity and presumed structural homology. The first 29 residues of IGFsare homologous to the B-chain of insulin (B region, 1-29), followed by12 residues that are analogous to the C-peptide of proinsulin (C region,30-41), and a 21-residue region that is homologous to the A-chain ofinsulin (A region, 42-62). The carboxy-terminal octapeptide (D region,63-70) has no counterpart in insulins and proinsulins (Murray-Rust, J.,et al., BioEssays 14 (1992) 325-331; Baxter, R. C., et al., J. Biol.Chem. 267 (1992) 60-65). The IGFs are the only members of the insulinsuperfamily in which the C region is not removed proteolytically aftertranslation. The 3D structure of insulin has been studied intensivelysince the first crystal structure determination in the 1960s (Adams, M.J., et al., Nature 224 (1969) 491-492). There are now structures ofinsulins in several oligomeric states, for insulins crystallized indifferent solvent conditions, and for many variants from differentspecies and chemical modifications. This is in stark contrast to IGFs(and proinsulins) for which no high definition structure has beenavailable prior to this report. Instead, the tertiary structure of IGF-Ihas been modeled after porcine insulin (Blundell, T. L., Proc. Natl.Acad. Sci. USA 75 (1978) 180-184). 2D NMR studies of IGF-I haveconfirmed that the solution structure is consistent with the model(Cooke, R. M., et al., Biochemistry 30 (1991) 5484-5491; Sato, A., etal., Int. J. Pept. Protein Res. 41 (1993) 433-440). However, NMR studiesof IGF-I have yielded structures only of low resolution, probablybecause IGF-I is soluble at the concentrations required for NMR only atpH values below 3 (Cooke, R. M., et al., Biochemistry 30 (1991)5484-5491; Sato, A., et al., Int. J. Pept. Protein Res. 41 (1993)433-440). More recently, better defined structures have been obtainedfor IGF-II (Terasawa, H., et al., EMBO J. 13 (1994) 5590-5597; Torres,A. M., et al., J. Mol. Biol. 248 (1995) 385-401) and for a Glu-3 to Argvariant of IGF-I (long-[Arg3]IGF-I) that additionally possesses a13-amino acid extension at the N-terminus (Laajoki, L. G., et al., J.Biol. Chem. 275 (2000) 10009-10015).

IGFBPs (insulin-like growth factor binding proteins -1 to -6) areproteins of 216 to 289 residues, with mature IGFBP-5 consisting of 252residues (Wetterau, L. A., et al., Mol. Gen. Metab. 68 (1999) 161-181).All IGFBPs share a common domain organization. The highest conservationis found in the N-(residues 1 to ca. 100) and C— (from residue 170)terminal cysteine rich regions. Twelve conserved cysteines are found inthe N-terminal domain and six in the C-terminal domain. The central,weakly conserved part (L-domain) contains most of the cleavage sites forspecific proteases (Chernausek, S. D., et al., J. Biol. Chem. 270 (1995)11377-11382). Several different fragments of IGFBPs have been describedand biochemically characterized so far (Mazerbourg, S., et al.,Endocrinology 140 (1999) 4175-4184). Mutagenesis studies suggest thatthe high affinity IGF binding site is located in the N-terminal domain(Wetterau, L. A., et al., Mol. Gen. Metab. 68 (1999) 161-181;Chernausek, S. D., et al., J. Biol. Chem. 270 (1995) 11377-11382) andthat at least IGFBP-3 and IGFBP-2 contain two binding determinants, onein the N— and one at the C-terminal domains (Wetterau, L. A., et al.,Mol. Gen. Metab. 68 (1999) 161-181). Recently, a group of IGFBP-relatedproteins (IGFBP-rPs) which bind IGFs with lower affinity than IGFBPshave been described (Hwa, V., et al., The IGF binding proteinsuperfamily, In: Rosenfeld, R. G., and Roberts, C. T. (eds.), The IGFsystem, Molecular Biology, Physiology, and Clinical Applications (1999),Humana Press, Totowa, pp. 315-327). IGFBPs and IGFBP-rPs share thehighly conserved and cysteine-rich N-terminal domain which appears to becrucial for several biological actions, including their binding to IGFsand high affinity binding to insulin (Hwa et al., 1999). N-terminalfragments of IGFBP-3, generated for example by plasma digestion, alsobind insulin and physiologically are thus likely relevant for insulinaction. Beyond the N-terminal domain, there is a lack of sequencesimilarity between the IGFBPs and IGFBP-rPs.

The sequences of human IGFBP-1 to −6 are described in detail in theSwissProt Database (http://www.expasy.ch) and identified by thefollowing Accession Nos.: Name Accession No. IGFBP-1 P 08833 IGFBP-2 P18065 IGFBP-3 P 17936 IGFBP-4 P 22692 IGFBP-5 P 24593 IGFBP-6 P 24592

The amino acid positions described in the following refer to thesequence of the mature forms the human IGF binding proteins (sequenceafter removal of the signaling peptide starts with amino acid inposition 1, see also Tables 1 to 6).

The association of insulin-like growth factors with neoplasia indicatesthat inhibition of the IGF signaling pathway in tumors might be asuccessful strategy in cancer therapy. Such modulation might beaccomplished, for example, through exogenous administration ofrecombinant inhibitory IGFBPs and effective fragments thereof.Additionally, tumor cell IGFBP production, inhibition or degradation maybe controlled by agents such as tamoxifen and ICI 182,780 that modifytumor IGFBP production (Khandwala, H. M., et al., Endocr. Rev. 21 (2000)215-244). The consequent alteration in IGFBP-3 levels appears in certaininstances to inhibit IGF-1-stimulated cell proliferation (Khwandala etal., 2000). There is also recent evidence that IGFBP-3 may be ap53-independent effector of apoptosis in breast cancer cells via itsmodulation of the Bax:Bcl-2 protein ratio (Butt, A. J., et al., J. Biol.Chem. 275 (2000) 39174-39181; Wetterau, L. A., et al., Mol. Gen. Metab.68 (1999) 161-181).

IGFBPs show a significant inhibition of tumor cell proliferation invitro but only very high doses result in inhibition of tumor growth invivo (van den Berg, C. L., et al., Eur. J. Cancer 33 (1997) 1108-1113).Van den Berg therefore covalently coupled IGFBP-1 to polyethyleneglycol, which leads to a prolonged serum half-life. However, theinhibitory effects of the pegylated IGFBP-1 is still not sufficient fortherapeutic intervention in humans because only partial response isobserved even if pegylated IGFBP-1 is given in doses of 1 mg/dose dailyin mice. This corresponds to a dose of 50 mg/kg×day which can not beadministered to humans by established procedures and can not be producedeconomically.

IGF releasing peptides are described by Loddick, S. A., et al., Proc.Natl. Acad. Sci. USA 95 (1998) 1894-1898 and Lowman, H. B., et al.,Biochemistry 37 (1998) 8870-8878. The described molecules which are ableto displace IGFs from their binding proteins are either mutants of IGF-Iwhich bind to IGFBPs but are not able to stimulate the IGF-R or a 14amino acid peptide with similar properties derived from a phage-displaylibrary. The biological activities of the peptides were shown byadministration either by injection into the lateral ventricle of thebrain or infused by a minipump.

Mutagenesis studies for IGFs indicated that IGF amino acid residues Glu3, Thr 4, Gln 15 and Phe 16 of IGF-I and Glu 6, Phe 48, Arg 49 and Ser50 in IGF-II are important for binding to IGFBPs (Baxter, R. C., et al.,J. Biol. Chem. 267 (1992) 60-65; Bach, L. A., et al., J. Biol. Chem. 268(1993) 9246-9254; Luethi, C., et al., Eur. J. Biochem. 205 (1992)483-490; Jansson, M., et al., Biochemistry 36 (1997) 4108-4117). Baxteret al. (1992) suggested that the IGF-I amino acid residues Glu 3, Thr 4,Gln 15 and Phe 16 are crucial for interaction with IGFBP-3, whereasresidues Phe 49, Arg 50 and Ser 51 are of secondary importance. It alsowas suggested that Phe 26 of IGF-II plays a role in changing the localstructures of IGFs but does not directly bind to IGFBPs (Terasawa, H.,et al., EMBO J. 13 (1994) 5590-5597).

Kalus, W., et al., in EMBO J. 17 (1998) 6558-6572, describe proteolyticstudies of human IGFBP-5 and the cloning and expressing of the domain ofIGFBP-5 between residues 40-92 (mini-IGFBP-5); this domain binds IGF-Iand IGF-II with KD values of 37 nM and 6 nM, respectively, as well asthe determination of the solution structure of uncomplexed mini-IGFBP-5by NMR. Kalus et al. identified some IGF binding sites which areresidues Val49, Tyr50, Pro62 and Lys68 to Leu75 of IGFBP-5.

Imai, Y., et al., in J. Biol. Chem. 275 (2000) 18188-18194, describe anIGFBP-3 variant and an IGFBP-5 variant, each with a five-foldsubstitution pattern at amino acid positions hypothesized by Kalus etal. as IGF binding sites. Imai et al. found that a substantialalteration of the amino acid residues simultaneously at positions 68,69, 70, 73 and 74 results in a 1000-fold or larger reduction in theaffinity for IGF-I in relation to the affinity of wild-type IGFBP-5.

Conover, C. A., et al., in J. Biol. Chem. 270 (1995) 4395-4400, describeprotease-resistant mutants of IGFBP-4. All four IGFBP-4 mutants aroundthe putative cleavage site at Met135-Lys136 and the wild-type proteinbind IGFs with equivalent affinities.

Byun, D., et al., in J. Endocrinology 169 (2001) 135-143, postulateseveral regions involved in IGF binding by IGFBP-4. Deletion of segmentsLeu72-Ser 91 or Leu72-His74 results in loss of IGF binding. Alsomutation of certain cysteine residues significantly reduces the bindingof IGFs.

Thus, these described mutant forms of insulin-like growth factor bindingproteins have reduced or equivalent affinities for IGF-I and/or IGF-II.Mutants of IGFBPs with a significantly higher affinity and a thereforeimproved effectiveness have not been known heretofore and there exists aneed for such molecules as well as for methods for identifying IGFBPantagonists.

SUMMARY OF THE INVENTION

The invention provides a crystal suitable for X-ray diffraction,comprising a complex of insulin-like growth factor I or II and apolypeptide consisting of the amino acids 39-91 of IGFBP-1, the aminoacids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the aminoacids 39-91 of IGFBP-4, the amino acids 40-92 of IGFBP-5, or the aminoacids 40-92 of IGFBP-6 or a fragment thereof consisting at least of the9^(th) to 12^(th) cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, orIGFBP-5 or at least of the 7^(th) to 10^(th) cysteine of IGFBP-6 (suchpolypeptides and fragments are hereinafter also referred to as“mini-IGFBPs).

Such a crystal is suitable for determining the atomic coordinates of thebinding sites of IGF-I, IGF-II, and IGFBPs, and therefore allows theoptimization of these molecules to identify and improve stabilizinginteractions between IGF-I or IGF-II and IGFBPs. Preferably, the crystaleffectively diffracts X-ray for the determination of the atomiccoordinates of said complex to a resolution of 1.5 to 3.5 Å. The crystalis arranged in the cubic space group P2,3 having unit cell dimensions of74.385 Å×74.385 Å×74.385 Å.

The invention further provides a method for producing a crystal suitablefor X-ray diffraction, comprising

-   (a) contacting IGF-I or IGF-II with a polypeptide consisting of the    amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the    amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the    amino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or    a fragment thereof consisting at least of the 9^(th) to 12^(th)    cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at    least of the 7^(th) to 10^(th) cysteine of IGFBP-6, to form a    complex which exhibits restricted conformation mobility, and-   (b) obtaining a crystal from the complex so formed suitable for    X-ray diffraction.

Using this crystal, the atomic coordinates of the complex can bedetermined.

The invention further comprises a method for identifying a mutant ofIGFBP or a mutant of a fragment thereof consisting at least of the9^(th) to 12^(th) cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, orIGFBP-5 or at least of the 7^(th) to 10^(th) cysteine of IGFBP-6, andhaving enhanced binding affinity for IGF-I and/or IGF-II comprising

-   (a) constructing a three-dimensional structure of the complex of    IGF-I or IGF-II and a polypeptide consisting of the amino acids    39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, the amino acids    47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, the amino acids    40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or a fragment    thereof consisting at least of the 9^(th) to 12^(th) cysteine of    IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the    7^(th) to 10^(th) cysteine of IGFBP-6, based on the atomic    coordinates of a crystal consisting of IGF-I or IGF-II and said    polypeptide;-   (b) employing said three-dimensional structure and modeling methods    to identify said mutant in which an amino acid residue within a    distance of 5 Å to a hydrophobic amino acid residue of IGF-I or    IGF-II is modified in that the hydrophobic interaction between IGF-I    or IGF-II and said mutant of IGFBP is enhanced;-   (c) producing said mutant;-   (d) assaying said mutant to determine said enhanced binding affinity    for IGF.

The invention further comprises a method for identifying a mutant ofIGFBP-5 with enhanced binding affinity for IGF-I, said method comprising

-   (a) constructing a three-dimensional structure of the complex of    IGF-1 and IGFBP-5 defined by the atomic coordinates shown in FIGS. 5    and 6;-   (b) employing said three-dimensional structure and modeling methods    to identify an amino acid residue in IGFBP-5 within a distance of 5    Å or shorter to an amino acid residue of IGF-I, wherein said residue    of IGFBP-5 can be modified hydrophobically in that the hydrophobic    interaction between IGF and IGFBP-5 is enhanced;-   (c) producing said mutant;-   (d) assaying said mutant to determine said enhanced binding affinity    for IGF.

The amino acid residue(s) in which IGFBP(s) is/are modified is/arepreferably selected from the amino acids 39-91 of IGFBP-1, the aminoacids 55-107 of IGFBP-2, the amino acids 47-99 of IGFBP-3, the aminoacids 39-91 of IGFBP-4, the amino acids 49-92 of IGFBP-5, or the aminoacids 40-92 of IGFBP-6.

Especially preferred IGFBP mutants are modified at amino acid residues49, 70 and/or 73 corresponding to IGFBP-5 sequence alignment andaccording to Table 7.

The invention therefore provides mutant IGFBPs (“IGFBPs” as used hereinmeans IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 and/or IGFBP-6) withenhanced affinity (preferably about 3-fold to 10-fold increased affinityto the corresponding wild-type IGFBP) for IGF (“IGF” as used hereinmeans IGF-I and/or IGF-II), improved inhibitory potency for the activityof IGF in vitro and in vivo and therefore improved therapeuticeffectiveness.

The invention further provides a method for identifying a compoundcapable of binding to IGFBP, comprising

-   (a) constructing a three-dimensional structure of a complex of IGF-I    or IGF-II and a polypeptide consisting of the amino acids 39-91 of    IGFBP-1, amino acids 55-107 of IGFBP-2, amino acids 47-99 of    IGFBP-3, amino acids 39-91 of IGFBP-4, amino acids 40-92 of IGFBP-5,    amino acids 40-92 of IGFBP-6 or a fragment thereof consisting at    least of the 9^(th) to 12^(th) cysteine of IGFBP-1, IGFBP-2,    IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7^(th) to 10^(th)    cysteine of IGFBP-6, based on the atomic coordinates of a crystal    consisting of IGF-I and said IGFBP;-   (b) employing said three-dimensional structure and modeling methods    to identify a compound forming a complex with said IGFBP by    hydrophobic binding with amino acids 49, 50, 70, 71 and 74 in the    case of IGFBP-5 and in the case of IGFBP-1, IGFBP-2, IGFBP-3,    IGFBP-4 and IGFBP-6 with corresponding amino acids according to    Table 7;-   (c) producing said compound;-   (d) determining the binding between the compound and IGFBP.

The invention further provides a method of inhibiting the binding of IGFto the IGFBP in a subject, preferably a human subject, comprisingadministering an effective amount of an above-described mutant of IGFBPto the subject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for co-crystallizing IGF-I orIGF-II with a truncated N-terminal fragment of IGFBP, preferably ofIGFBP-5 (mini-IGF), where the crystals diffract X-rays with sufficientlyhigh resolution to allow determination of the three-dimensionalstructure of said complex, including atomic coordinates. Thethree-dimensional structure (e.g. as provided on computer-readablemedia) is useful for rational drug design of IGFBP mutants with modifiedaffinity for IGF-I or IGF-II, preferably with an improved affinity.There is specifically provided a method for co-crystallizing IGF-I witha polypeptide consisting of an isolated folded domain of IGFBPs(mini-IGFBPs), which is formed by the amino acids between the 9^(th) andthe 12^(th) cysteine of IGFBP-1 to IGFBP-5 or the 7^(th) and 10thcysteine of IGFBP-6 and additionally including up to 7 amino acidsN-terminal of this fragment and up to 5-20 amino acids C-terminal tothis fragment. The amino acids 39-91 of BP-1, the amino acids 55-107 ofIGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 ofIGFBP-4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 ofIGFBP-6 or fragments thereof are especially suitable to form a complexwith IGF-I or IGF-II which exhibits restricted conformational mobilityand high suitability for X-ray diffraction.

Such a complex co-crystallizes in a manner sufficient for thedetermination of atomic coordinates by X-ray diffraction. The crystaleffectively diffracts X-ray for the determination of the atomiccoordinates of the complex to a resolution of 1.5 or at least better(less) than 3.5 Å. Said IGFBP fragments are able to form a compact andglobular structure whose scaffold is secured by an inside packing of twocysteine bridges and stabilized further by a three-stranded β-sheet. Thefolded fragments are still able to bind IGF-I and IGF-II with highaffinities. Other forms of the IGFBPs such as full-length IGFBPs, theisolated C-terminal domain of IGFBPs or fragments without N-terminaltruncation do not co-crystallize with IGF in a suitable manner forX-ray-based determination of the structure at high resolution.

Knowledge of the crystal structure enables the production of specificIGFBP mutants which develop improved interaction with, therebyexhibiting enhanced affinity for, IGF and, as a consequence, haveimproved therapeutic efficacy as IGF antagonists. Such IGFBP mutantswith increased affinity for IGF are capable of preventing the formationof the complex between naturally occurring IGF and IGF-I receptor(IGF-R) in vitro and in vivo and, thereby, of effecting an decrease inthe concentration of biologically active, free IGF. Such rationaldesigned IGF antagonists are therefore capable of inhibiting tumorgrowth and inducing apoptosis in tumor cells more efficient than naturalIGFBPs. As a result, lower doses of the optimal designed IGFBP mutantswith enhanced affinity are needed for achieving an effect comparable tothat of naturally occurring IGFBPs.

A further embodiment of the invention is the identification andoptimization of non-proteinaceous compounds which bind to the IGFbinding site of IGFBPs and prevent the formation of an inhibitorycomplex between IGFs and IGFBPs and therefore activates the anabolicaction of IGF. Such “IGF-releasing compounds” can be identifiedaccording to the invention on the basis of the crystal data, usingprotein-ligand docking programs such as FlexX (Kramer, B., et al.,Proteins: Structure, Functions and Genetics 37 (1999) 228-241).

The X-ray diffraction patterns of the invention have a sufficiently highresolution to be useful for three-dimensional modeling of an IGFreleasing compound. Preferably, the resolution is in the range of 1.5 to3.5 Å, preferably 1.5 to 3.0 Å. Three-dimensional modeling is performedusing the diffraction coordinates from these X-ray diffraction patterns.The coordinates are entered into one or more computer programs formolecular modeling as known in the art. Such molecular modeling canutilize known X-ray diffraction molecular modeling algorithms ormolecular modeling software to generate atomic coordinates correspondingto the three-dimensional structure of at least one IGF releasingcompound.

Such a compound shows affinity for IGFBP based on stereochemicalcomplementary relative to naturally occurring IGFs. Such stereochemicalcomplementary according to the present invention is characterized by amolecule that matches intra-site surface residues that form the contoursof IGFBPs as enumerated by the coordinates set out in FIGS. 5 and 6. Theresidues that define the contours are depicted in FIGS. 5 and 6.Matching according to the invention means that the identified atoms oratom groups interact with the IGFBP surface residues, for example viahydrogen bonding or by enthalpy-reduced van der Waals interactions whichprevent or reduce the interaction between IGFBP and IGFs and therebypromote the release of the biologically active compound from the bindingsite. In general, the design of a molecule possessing stereochemicalcomplementary to the contours of IGFBPs can be accomplished by means oftechniques that optimize either chemically or geometrically the fitbetween a molecule and a target receptor. Known techniques of this sortare reviewed by Sheridan, R. P., and Venktaraghavan, R., Acc. Chem. Res.20 (1987) 322; Goodford, P. J., J. Med. Chem. 27 (1984) 557; Verlinde,C., and Hol, W., Structure 2 (1994) 577; and Blundell, T. L. et al.,Nature 326 (1987) 347. The design of optimized IGFBP ligands based onthe invention is preferably done by the use of software such as GRID(Goodford, P. J., J. Med. Chem. 28 (1985) 849-857), a program thatdetermines probable interaction sites between probes with variousfunctional group characteristics and the protein surface—is used toanalyze the surface sites to determine structures of similar inhibitingproteins or compounds.

The program DOCK (Kuntz, I. D., et al., J. Mol. Biol. 161 (1982)269-288) can also be used to analyze an active site or ligand bindingsite and suggest ligands with complementary steric properties. Severalmethodologies for searching three-dimensional databases to testpharmacophore hypotheses and select compounds for screening areavailable. These include the program CAVEAT (Bacon et al., J. Mol. Biol.225 (1992) 849-858) which uses databases of cyclic compounds which canact as spacers to connect any number of chemical fragments alreadypositioned in the active site. The program LUDI (Bohm, H. J., et al., J.Comput. Aided Mol. Des. 6 (1992) 61-78 and 593-606) defines interactionsites into which both hydrogen bonding and hydrophobic fragments fit.

Programs suitable for searching three-dimensional databases to identifyalso non-proteinaceous molecules bearing a desired pharmacophoreinclude: MACCS-3D and ISIS/3D (Molecular Design Ltd., San Leandro,Calif.), ChemDBS-3D (Chemical Design Ltd., Oxford, U.K.), and Sybyl/3 DBUnity (Tripos Associates, St. Louis, Mo.).

Programs suitable for pharmacophore selection and design include: DISCO(Abbott Laboratories, Abbott Park, Ill.), Catalyst (Bio-CAD Corp.,Mountain View, Calif.), and ChemDBS-3D (Chemical Design Ltd., Oxford,U.K.).

Databases of chemical structures are available from a number of sourcesincluding Cambridge Crystallographic Data Centre (Cambridge, U.K.) andChemical Abstracts Service (Columbus, Ohio).

De novo design programs include Ludi (Biosyrn Technologies Inc., SanDiego, Calif.), Sybyl (Tripos Associates) and Aladdin (Daylight ChemicalInformation Systems, Irvine, Calif.).

Those skilled in the art will recognize that the design of suchcompounds may require slight structural alteration or adjustment of achemical structure designed or identified using the methods of theinvention.

Non-proteinaceous compounds and IGFBP mutants with increased bindingaffinity for IGF can be identified by incubating said compounds ormutants with an IGF-I/IGFBP-5 complex and measuring the binding ofreleased IGF-I to IGF-I receptor expressing cells. Due to the binding ofIGF-I to its cell-bound receptor, the receptor is activated andautophosphorylated. Alternatively, radiolabeled IGF-I can be used andits binding to its receptor after release from the complex can bedetermined.

Formation of the IGF-1 mini-IGFBP-5 complex buries a binding surfacetotalling about 550 Å². Of the eleven IGFBP-5 amino acid residues within5 Å of IGF, six are hydrophobic, three of which are surface-exposedleucines and valines and are of primary importance for hydrophobicinteraction to IGFs (FIGS. 1 to 4). On the IGF side, four of the elevenamino acid residues within 5 Å of mini-IBFBP-5 are hydrophobic (FIGS. 1to 4).

The IGFBPs bind to IGF-I and IGF-II by presenting a binding surfacecomplementary to that of IGF. The IGF binding surface consists of arelatively flat hydrophobic surface, a small hydrophobic depression, twohydrophobic protruberances, and surrounding polar residues.Identification of the IGF binding surface itself (FIG. 3) enables thedesign of binding partners in general, and optimization of known bindingpartners in particular. General binding partners will have at least twoof the following features 1 to 4:

-   1. Non-polar atoms lying approximately in a plane defined by atoms    Leu74 CD1 and CD2, Val49 CG1 and CG2, Leu70 CB, and Tyr 50 CB,    within a perimeter defined by IGF residues Glu9, Glu3, Leu54, Phe 16    and by BP5 atom Tyr 50 OH and depicted in FIG. 3 such that they    present an approximately planar and hydrophobic molecular surface of    at least 20 square Angstroms.-   2. A non-polar atom or atoms near the positions of Leu 70 CG, CD1    relative to IGF, filling the depression of IGF as seen in the    complex structure.-   3. Hydrophobic and/or aromatic interactions with the side chains of    Phe16, Val17, and/or Leu54 of IGF as defined by a net buried surface    area in the complex of at least 20 square Angstroms.-   4. Polar (hydrogen bonding and/or charge complementary)    interactions, either directly or via bridging solvent molecules,    with one or more of the following IGF atoms: Asp12 OD1,2; Glu9    OE1,2; Glu3OE1,2; Glu58OE1,2; Thr4 O,OG1; Cys52O; Ser51 OG;    Asp53OD1,2; Arg55NH1,2,NE; Arg21NH1,2,NE; Val17O; Cys18O;    Asp20OD1,2,N; Gln15O,OD1,ND2.

Abbreviations: Letters corresponding to standard amino acid atom naming(according to the International Union of Physicists andChemists-IUPAC-naming). CG: Carbon Cγ CB: Carbon Cβ OE: Oxygen Oε OH:Oxygen Oη OD: Oxygen Oδ O: Backbone Oxygen NH: Nitrogen Nη NE: NitrogenNε N: Backbone Nitrogen ND: Nitrogen Nδ

The principal IGF/IGFBP interaction, shown in the example of IGF-1mini-IGFBP-5 interaction, is a hydrophobic sandwich that consists ofinterlaced protruding side chains of IGF-I and solvent exposedhydrophobic side chains of the mini-IGFBP-5 (FIGS. 1 to 4). Theside-chains of IGF-I Phe 16, Leu 54 and also Glu 3, are inserted deepinto a cleft on the mini-IGFBP-5 (FIGS. 1 to 4). This cleft is formed byside chains of Arg 53, Arg 59 on the solvent exposed side of themolecule and by Val 49, Leu 70, Leu 74 on the opposite inner side, witha base formed by residues Cys 60 and Leu 61. Phe 16 makes directcontacts with the backbone and side chain of Val 49, and with Cys 60 ofmini-IGFBP-5. The hydrophobic cluster is closed on the solvent side byside chains of Glu 3 and Glu 9 of IGF-I and His 71 and Tyr 50 ofmini-IGFBP-5. These residues form a network of hydrogen bonds; inaddition Arg 59 of mini-IGFBP-5 makes hydrogen bonds with Glu 58 (FIGS.2 to 4).

Arg 53 and Arg 59 of mini-IGFBP-5 isolate the hydrophobic sandwich fromthe solvent close to the C-terminus. In the full length IGFBP-5, thesegment corresponding to the C-terminus of mini-IGFBP-5 is followed bynine hydrophilic residues and then by at least 30 residues of mixedtypes. Thus, the conformations seen in the structure of the complex nearthe C-terminus of mini-IGFBP-5 are likely to be preserved in the complexof IGF-I with the full length-IGFBP-5. The mini-IGFBP-5 domain beginspreferably at residue 40 of full length IGFBP-5.

The hydrophobic residues Val 49, Leu 70 and Leu 73 of IGFBP-5 arecrucial for binding to IGFs. Since these residues are highly conservedamong all IGFBPs, these hydrophobic interactions dominate the IGFbinding properties of all IGFBPs.

The increased inhibitory potency of the mutant IGFBPs and fragmentsthereof results in the inhibition of the binding to andautophosphorylation of the IGF-R (as described by Kalus, W., et al., inEMBO J. 17 (1998) 6558-6572) at significantly lower concentrations thanobserved for the wildtype proteins and the corresponding fragments. Thehigher potency of the mutant IGFBPs furthermore can be shown by theinhibition of the growth of tumor cells in vitro and in vivo. The growthof several tumor cell lines is known to be significantly reduced byIGFBPs. IGFBP-1 for example inhibits the growth of MCF-7 and MDA-MB-435Acells in vitro and the growth of tumors formed MDA-MB-231 cells in vivoin mice (van den Berg, C. L., et al., Eur. J. Cancer 33 (1997)1108-1113). IGFBP mutants with increased affinity inhibit the growth ofthese tumor cells at lower concentrations than the wild type proteins.

The following mutations of IGFBPs are preferred for enhancing bindingaffinity to IGF (numbering according to IGF-BP5 as aligned in FIG. 1)(standard one-letter abbreviation for amino acids used): TABLE 1 IGFBP-1Amino Original acid No. amino acid Preferred mutations¹⁾ 48 V L, I , M,F, Y, W 49 A Y , R, K 52 R W, Y , M, F, H 60 R Y, W, F 69 L Y, W, M, I,F 72 L I, Y, W, M, F 73 T V, L, Y, W, M, I, F 74 R H, D 82 E R, K, H, N,Q, S, T, A, G

TABLE 2 IGFBP-2 Amino Original acid No. amino acid Preferred mutations¹⁾64 V L, I , M, F, Y, W 65 Y R, K 68 R W, Y , M, F, H 76 Y W, F 85 L Y,W, M, I, F 86 Q T, S, R, K, N, H , Y, C 88 L I, Y , W, M, F 89 V L, I,Y, W, M, F 90 M H, D

TABLE 3 IGFBP-3 Amino Original acid No. amino acid Preferred mutations¹⁾56 I L , V, M, F, Y, W 57 Y R, K 60 R W, Y , M, F, H 68 Q L, Y, W, F 75R Q 77 L Y, W, M, I, F 78 Q T,S, R, K, N, H , Y, C 80 L I, Y, W, M , F81 L Y, W, M, I, F

TABLE 4 IGFBP-4 Amino Original acid No. amino acid Preferred mutations¹⁾48 V L, I , M, F, Y, W 49 Y R, K 52 R W, Y, M, F, H 60 Y W, F 67 K Q 69L Y, W, M, I, F 72 L I, Y , W, M, F 73 M Y, W, I, F 74 H D

TABLE 5 IGFBP-5 Amino Original acid No. amino acid Preferred mutations¹⁾49 V L, I , M, F, Y, W 50 Y R, K 53 R W, Y , M, F, H 61 L Y, W, F 68 K Q70 L Y, W, M, I, F 73 L I, Y, W , M, F 74 L Y, W, M, I, F 75 H D 83 E R,K, H, N, Q, S, T, A, G

TABLE 6 IGFBP-6 Amino Original acid No. amino acid Preferred mutations¹⁾49 V L, I , M, F, Y, W 50 Y R, K 53 N R, W, Y , M, F, H 61 H L, Y, W, F68 A K, Q 70 L Y, W, M, I, F 71 R T, S, H , K, N, Q, Y, C 73 L I, Y, W ,M, I, F 74 L Y, W, M, I, F 75 L H, D¹⁾Amino acids are given in the standard one-letter amino acid code andare to be understood as alternative amino acid exchanges (or). Forinstance, the last line of Table 6 means that amino acid residue 75 ofIGFBP-6, which is leucine (L), can# preferably be modified to be either histidine (H) or aspartic acid(D). Table 6 is additionally to be interpreted such that amino acids 49,50, 53, 61, 68, 70, 73, 74 and/or 75 can be exchanged in order toimprove affinity. Especially preferred are # IGFBP mutants with singlepoint mutations. Most preferred are IGFBP mutants having a single pointmutation from the bold face residues. This applies correspondingly tothe other tables.

TABLE 7 Sequence alignment showing corresponding amino acids of IGFBP-1to -6 Amino Acid No. IGFBP-1 IGFBP-2 IGFBP-3 IGFBP-4 IGFBP-5 IGFBP-6 4864 56 48 49 49 49 65 57 49 50 50 52 68 60 52 53 53 60 76 68 60 61 61 6783 75 67 68 68 69 85 77 69 70 70 70 86 78 70 71 71 72 88 80 72 73 73 7389 81 73 74 74 74 90 82 74 75 75

The presented structure enables in silico screens for small IGFBP ligandinhibitors with the potential to release “free” bioactive IGF.Displacement of IGF from their binding proteins are therapeuticallyuseful in treating a variety of potential indications, including shortstature, renal failure, type I and type II diabetis, stroke and otherneuro-degenerative diseases.

The compounds and IGFBP mutants of the present invention can beformulated according to methods for the preparation of compositions,preferably pharmaceutical compositions, which methods are known to theperson skilled in the art. Preferably, such a compound and IGFBP mutantis combined in a mixture with a pharmaceutically acceptable carrier.Such acceptable carriers are described in, for example, Remington'sPharmaceutical Sciences, 18^(th) ed., 1990, Mack Publishing Company,edited by Oslo et al. (e.g. pp. 1435-1712). Typical compositions containan effective amount of a non-proteinaceous compound or IGFBP mutantaccording to the invention, for example from about 1 to 10 mg/ml,together with a suitable amount of a carrier. The compounds and IGFBPmutants may be administered preferably parenterally.

The invention further provides pharmaceutical compositions containing anon-proteinaceous compound or IGFBP mutant according to the invention.Such pharmaceutical compositions contain an effective amount of acompound and IGFBP mutant of the invention, together withpharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents of various buffer contents (e.g., acetate, phosphate,phosphate-buffered saline), pH and ionic strength, additives such asdetergents and solubilizing agents (e.g., Tween®80, polysorbate,Pluronic®F68), antioxidants (e.g., ascorbic acid, sodium metabisulfite),preservatives (Timersol®, benzyl alcohol) and bulking substances (e.g.,saccharose, mannitol).

Compositions and pharmaceutical compositions according to the inventionare manufactured in that the substances in pure lyophilized form aredissolved at a concentration up to from 1 to 20 mg/l in PBS orphysiological sodium chloride solution at a neutral pH value. For bettersolubility it is preferred to add a detergent.

Typically, in a standard cancer treatment regimen, patients are treatedwith dosages in the range of between 0.5 to 10 mg substance/kg weightper day.

The following examples, references, sequence listing and figures areprovided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1A: Sequence alignment of IGF-I and IGF-II. Bold underlinedresidues of IGF-I make contacts with mini-IGFPB5. Residues responsiblefor binding to the IGF-I receptor (IGF-R) are marked with an asteriskabove the sequence.

FIG. 1B: Multiple sequence alignment of the N-terminal domains of humanIGF-BPs 1-6. The mini-BP construct, numbered according to BP5 numbering,is marked above the aligned residues with “m”, including GS whichindicate additional residues from the cloning vector. (After position81, mini-BP5 was disordered in the X-ray structure; this is indicatedwith italics.) BP5 residues that interact with IGF-I are shownunderlined and in bold face. The degree of conservation of the residuesis marked under the alignment with * for strict conservation, : forstrict conservation of residue type, and . for relatively highconservation. The consensus sequence uses the following code to depictlevel of strict conservation: o alcohol, l aliphatic, a aromatic, ccharged, h hydrophobic, −negative, p polar, +positive, s small, u tiny,t turnlike).

FIG. 2: The overall structure of the IGF-I (tube model) mini-IGFBP5(molecular surface) complex. Side chains plotted show the IGF residuesin contact with BP5. Particularly important is Phe16, seen filling ahydrophobic depression on the BP5 surface.

FIG. 3: Similar to FIG. 2, whereby the IGF is depicted with itsmolecular surface and BP5 is depicted as a tube model. Side chains ofBP5 responsible for binding to IGF are also depicted. The surface of IGFPhe16 is prominent, as is the relatively flat hydrophobic IGF surfacecontributing to the interface.

FIGS. 4A and 4B: Summary of IGF-BP5 and IGF-I contacts. Interactionscontributing to the binding affinity consist of hydrophobic interactions(a) (involving especially residues Leucines 70, 73, and 74 of BP5 andPhe16 of IGF-I) and also polar interactions (b). Enhancement of BP-IGFbinding relies especially on the enhancement of hydrophobicinteractions, either by increasing the intermolecular contact surfacewith these or with additional residues, or by the introduction offurther polar contacts.

-   -   (A) Packing contacts between IGFBP-5 and IGF-I. Contacts are        denoted according to nearest distances, whereby the closest        contacts include polar interactions.    -   (B) Polar contacts between IGFBP-5 and IGF-I. Abbreviations        denote hydrogen bonds (HB), CH—O hydrogen bonds (CHB), salt        bridge (SB), and side chain (SC) or main chain (MC)        interactions.

FIG. 5: Atomic coordinates of IGF-I in the complex with mini-IGFBP-5.

FIG. 6: Atomic coordinates of mini-IGFBP-5 in the complex with IGF-I.

FIG. 7: Binding of radioactive J-125 IGF-I to NIH 3T3 cells expressingthe IGF-R in the absence and in the presence of IGFBP-5 and compoundspotentially interfering with complex formation between IGF-I and IGFBP-5

FIG. 8: IGF-I induced autophosphorylation of the IGF-R expressed by NIH3T3 cells in the absence and in the presence of IGFBP-5 and compoundspotentially interfering with complex formation between IGF-I and IGFBP-5Sequence Listing SEQ ID NO:1 Primer FBP5LY. SEQ ID NO:2 Primer RBP5LY.SEQ ID NO:3 Primer FBP5LM. SEQ ID NO:4 Primer RBP5LM. SEQ ID NO:5 PrimerIBP4NdeI. SEQ ID NO:6 Primer IBP4BamHI. SEQ ID NO:7 Peptide GSALA. SEQID NO:8 Peptide GSHMDEAIH.

EXAMPLE 1

Crystallization, Data Collection and Derivatization

Mini-IGFBP-5 was produced as described by Kalus, W., et al., in EMBO J.17 (1998) 6558-6572 and in Example 6, and IGF-I was obtained fromOvoPepi, Australia. Crystallization was successful with 10% JeffamineM-600, 0.1 M sodium citrate, 0.01 M ferric chloride, pH 5.6. Within 11days, crystals appeared at 4° C., growing to a final size of about0.3×0.2×0.2 mm3. They belong to the cubic space group P213 and have unitcell dimensions a, b, c=74.385 Å, with one complex molecule perasymmetric unit. Soaking in precipitation buffer with heavy atomcompounds yielded a derivative K2PtCl4 (2.7 mM, 3 d) which wasinterpretable. All diffraction data were collected using a 300 mm MARResearch (Hamburg, Germany) image plate detector mounted on a Rigaku(Tokyo, Japan) RU300 rotating anode X-ray generator with graphitemonochromatized CuKα radiation. All image plate data were processed withMOSFLM (Leslie, A. G. W., Molecular Data in Processing, in: Moras, D.,Podjarny, A. D., and Thierry, J. C. (eds.), Crystallographic Computing 5(1991), Oxford University Press, Oxford, UK, pp. 50-61) and the CCP4program suite (Collaborative Computational Project, Number 4 1994).

EXAMPLE 2

Phase Calculation, Model Building and Refinement

The structure of the IGF/mini-IGFBP-5 complex was solved by the singleisomorphous replacement (s.i.r.) method using one heavy atom derivativedescribed above. Derivative data was analyzed with the native data set,first using isomorphous difference Patterson maps and employing thePatterson vector superposition methods implemented in SHELX-97(Sheldrick, G., Tutorial on automated Patterson interpretation to findheavy atoms, in: Moras, D., Podjarny, A. D., and Thierry, J. C. (eds.),Crystallographic Computing 5 (1991), Oxford University Press, Oxford,UK, pp. 145-157). The 2 heavy sites locations were confirmed bydifference Fourier methods with appropriate initial single site s.i.r.phases using CCP4 programs. The refinement of heavy atom parameters andcalculation of s.i.r. phases were done with SHARP (de la Fortelle, E.,and de Bricogne, G., Methods Enzymol. 276 (1997) 472-494). The finalparameters are given in Table 8. The initial s.i.r. phases were improvedwith SOLOMON (Abrahams, J. P., and Leslie, A. G. W., Acta. Cryst. D52(1996) 30-42) using an solvent fraction of 45%, resulting in a 2.1 Åelectron density map that was interpretable. Refinement was performed byconjugate gradient and simulated annealing protocols as implemented inCNS 1.0 (Brünger, A. T., et al., Acta Crystallogr. D54 (1998) 905-921.All protocols included refinement of individual isotropic B-factors andusing the amplitude based maximum likelihood target function. TheR-factor dropped to 21.8% (Rfree=26.2%, resolution range 16.2-2.1 Å) forthe final model including 47 water molecules. The water model wascalculated using ARP and verified by visual inspection. The finalrefinement statistics are shown in Table 8. TABLE 8 Statistics from thecrystallographic analysis native K₂PtCl₄ Resolution (Å) 16.2-2.118.6-2.5 Measurements 45345 32833 Unique measurements  8035 4925 %Complete (last shell/Å) 99.3 (96.9/2.23-2.11) 99.9 (95.4/2.64-2.5)R_(sym) (%) (last shell) 8.2 (44.8) 8.8 (49.5) R_(Cullis-iso) — 0.77P_(iso) — 1.48 Res. for phase calc. (Å) — 18.6-2.5 Mean FOM — 0.41Refinement statistics: Resolution range (Å) 16.2-2.1 reflections inworking set 7522 reflections in test set 501 R_(cryst) (%) 21.8 R_(free)(%) 26.2 Protein atoms (non-H) 765 Solvent atoms (non-H) 47 AverageB-factor (Å²) 38.1 r.m.s. ΔB (2 Å cutoff) 3.4 Deviations from ideality(r.m.s.): Bond lengths (Å) 0.013 Bond angles (°) 1.7

$R_{sym} = \frac{\sum{{{I(h)}_{i} - \left\langle {I(h)} \right\rangle}}}{\sum\left\langle {I(h)} \right\rangle}$

-   -   R_(Cullis-iso)=r.m.s. lack of closure/r.m.s isomorphous        difference    -   P_(iso)(Phasing power)=        |F_(H)|        /r.m.s. lack of closure for all reflections    -   Mean FOM=mean figure of merit    -   R_(cryst)=Crystallographic R-factor for reflections used in        refinement    -   R_(free)=Crystallographic R-factor for reflections not used in        refinement    -   r.m.s.=Root mean square

EXAMPLE 3

Determination of the Binding Affinity of IGFBP Mutants

The IGF-binding properties of wildtype and mutant fragments andfull-length IGFBPs were quantitatively analyzed by BIAcore biosensormeasurements. BIAcore 2000, Sensor Chip SA and HBS were obtained fromBIAcore AB (Uppsala, Sweden). All experiments were performed at 25° C.and HBS (20 mM HEPES, 150 mM NaCl, 3 mM EDTA, pH 7.5) was used as arunning buffer and for the dilution of ligands and analytes.Biotinylated IGF-I was immobilized at a concentration of 5 nM and 10 nMin HBS at a flow rate of 5 μl/min to the strepavidin coated sensor chipresulting in signals of 40 and 110 resonance units (RU). BiotinylatedIGF-II was immobilized at a concentration of 5 nM in HBS resulting in asignal of 20 RU. An empty flow cell was used as control for unspecificbinding and bulk effects. The low ligand concentration was necessary tolimit mass transport limitations and rebinding. For the same reason allkinetic experiments were performed at the highest possible flow rate of100 μl/min. Each protein (wildtype and mutant IGFBPs or fragments ofthese proteins) was injected at four concentrations (250, 50, 10, and 2nM). Each sample was injected for 2 min followed by dissociation inbuffer flow for 4 min. After the dissociation phase the sensor chip wasregenerated by injection of 10 μl 100 mM HCl at a flow rate of 5 μl/min.The kinetic parameters were calculated using the BIA evaluation 3.0software (BIAcore AB). After subtraction of the blank sensorgram thekinetic rate constants were calculated from a general fit of an overlayof the sensorgrams of all concentration of one analyte using the methodcalled “1:1 binding with mass transfer”. IGF-I and IGF-II werebiotinylated with a five-fold molar excess of D-biotinyl-E-aminocaproicacid-N-hydroxysuccinimide ester using the reagents and the operationinstructions of the Biotin Protein Labelling Kit (Roche DiagnosticsGmbH, DE). After blocking with lysine, the reaction was dialyzed against50 mM Na-phosphate, 50 mM NaCl, pH 7.5.

EXAMPLE 4

Inhibition of IGF-1-Induced IGF-R Phosphorylation by IGFBP Mutants

Confluent monolayers of NIH3T3 cells stably expressing human IGF-R in3.5 cm dishes were starved in DMEM containing 0.5% dialyzed fetal calfserum. After 48 h, cells were incubated without any hormone or with5×10⁻⁹ M IGF-1 or 1×10-8 M IGF-II; each sample was preincubated withincreasing concentrations of different IGF-binding proteins or fragmentsthereof at room temperature for 1 h. After a 10 min stimulation at 37°C., the medium was removed and cells were lysed with 250 μl of lysingbuffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Nonidet P40,1.5 mM MgCl₂, 1 mM EGTA (ethyleneglycol-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid, Aldrich, USA), 10mM sodium orthovanadate, and protease inhibitor cocktail Complete (RocheDiagnostics GmbH, DE) for 10 min on ice. Subsequently, cells werescraped off the plate and the insoluble material was separated bycentrifugation for 20 min at 4° C. The protein concentration of thesupernatant was determined using the BCA kit from Pierce, Rockford, USAaccording to the manufacturer's instructions. Equal proteinconcentration was incubated with the SDS sample buffer (63 mM Tris-HCl,pH 6.8, 3% SDS, 10% glycerol, 0.05% bromphenolblue, 100 mM DTT), boiledfor 5 min and loaded on a 7.5% SDS polyacrylamide gel. Afterelectrophoresis the proteins were transferred on a nitrocellulosemembrane which first was blocked for 1 h with the 3% BSA containing PBST(phosphate buffered saline-Tween®), then overnight incubated with 1μg/ml monoclonal anti-phosphotyrosine antibody 3-365-10 (RocheDiagnostics GmbH, DE) in PBST that contained 3% BSA. Unbound antibodywas removed by extensive washing. The blot was then incubated with1:10000 diluted anti-mouse IgG-specific antibody conjugated with horseraddish peroxidase (Roche Diagnostics GmbH, DE). The immunoblot wasdeveloped using the ECL kit from Amersham and the concentration of IGFBPwhich results in 50% inhibition of the IGF-I receptor phosphorylationwas determined.

EXAMPLE 5

Determination of the Inhibition of Tumor Cell Growth by IGFBP Mutants

MCF-7 cells (from ATCC, American type Culture Collection, Rockville,Md., U.S.A., HTB22) were used to investigate the inhibitory effect ofIGFBP mutants on tumor cells. 1000 MCF-7 cells were seeded per well inmedium containing 2.5% FBS (fetal bovine serum). The cells were culturedin the presence of various concentrations of IGFBPs for 48 h. Thepercentage of surviving cells was determined by MTT((3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay andthe concentration of binding protein which results in reduction of cellsurvival by 50% was determined.

EXAMPLE 6

Mutagenesis, Expression and Purification of Mini-IGFBP-5s and Subcloningof IGFBP-4 into Pet-28a (+)

6.1 Buffers and Media Cell growth media: LB-medium per 1 liter: peptone10 g, yeast extract 5 g, NaCl 10 g, adjusted to pH 7. LB-agar per 1liter: peptone 10 g, yeast extract 5 g, NaCl 10 g, bacto agar 15 g,adjusted to pH 7. Minimal per 1 liter: 0.5 g NaCl, 1 g citric acidmonohydrate, 36 mg medium ferrous citrate (pre-dissolved in conc. HCl),4.02 g KH₂PO₄, 7.82 g K₂HPO₄, 1 g ¹⁵N-NH₄Cl, 1.3 ml trace elementssolution (per liter of the stock solution: 2.5 g H₃BO₃, 2.0 g CoCl₂,1.13 g CuCl₂, 9.8 g MnCl₂, 2.0 g Na₂MoO₄), 1 ml Zn-EDTA solution (per mlof the stock solution: 5 mg EDTA, 8.4 mg zinc acetate), adjusted to pH7, autoclaved. Added afterwards: 25 ml autoclaved 20% (w/v) glucose, 560μl sterile filtered 1% (w/v) thiamine, 2 ml 1M MgSO₄. Antibiotic stocks:Ampicillin 50 mg/ml in dist. water, 0.22 μm filtrated, stored at −20° C.Kanamycin 25 mg/ml in dist. water, 0.22 μm filtrated, stored at −20° C.Chloram- 35 mg/ml in 96% ethanol, stored at −20° C. phenicol

Agarose-gel electrophoresis: TAE-buffer (50x) 2 M Tris-HCl (pH 8.0), 2 Mglacial acetic acid and 50 mM EDTA. Loading buffer (3x) 0.13%bromophenol blue, 0.13% xylene cyanol, 30% glycerol. Et-Br-solution 10mg/ml ethidiumbromide in dd H₂O. SDS-PAGE: Sample buffer (5x) 125 mMTris-HCl (pH 6.8), 10% SDS, 760 mM 2- mercaptoethanol, 0.13% bromophenolblue, 50% glycerol and 2 mM EDTA. Staining solution 0.125% CBB-R250 in500 ml 96% ethanol and 500 ml 10% acetic acid. Distaining solution 96%ethanol, 10% acetic acid and dest. H₂O in 4:3:3 proportion. Tricinegels: Cathode (top) 1 M Tris-HCl (pH 8.25), 1 M Tricine and 1% SDS.running buffer (10x) Anode (bottom) 2 M Tris-HCl (pH 8.9). runningbuffer (10x) Separation buffer 3 M Tris-HCl (pH 8.9) and 0.3% SDS.Stacking buffer 1 M Tris-HCl (pH 6.8) and 0.3% SDS. Separationacrylamide 48% (w/v) acrylamide, 1.5% (w/v) N,N′-methylene-bis-acrylamide. Stacking acrylamide 30% (w/v) acrylamide, 0.8% (w/v)N,N′-methylene-bis- acrylamide. APS 10% ammonium persulphate in dd H₂O.Separation gel (main) for 2 70 × 80 × 0.75 mm mini-gels: 1.675 ml H₂O,2.5 ml separation buffer, 2.5 ml separation acrylamide, 0.8 ml glycerol,25 μl APS and 2.5 μl TEMED. Separation gel 1.725 ml H₂O, 1.25 mlseparation buffer, o.75 ml separation (intermediate) acrylamide, 12.5 μlAPS and 1.25 μl TEMED. Stacking gel 2.575 ml H₂O, 0.475 ml stackingbuffer, 0.625 ml stacking acrylamide, 12.5 μl 0.5 M EDTA (pH 8.0), 37.5μl APS and 1.9 μl TEMED. Protein purification: Buffer A 6 Mguanidinium-HCl, 100 mM NaH₂PO₄, 10 mM Tris and 10 mM 2-mercaptoethanol,pH 8.0. Buffer B 6 M guanidinium-HCl, 100 mM NaH₂PO₄, 10 mM Tris and 10mM 2-mercaptoethanol, pH 6.5 Buffer C 6 M guanidinium-HCl, 100 mMNa-acetate and 10 mM 2- mercaptoethanol, pH 4.0. Buffer D 6 Mguanidinium-HCl, pH 3.0. Buffer E 200 mM arginine, 1 mM EDTA, 100 mMTris-HCl, 2 mM reduced glutathione, 2 mM oxidised glutathione, pH 8.4.PB(0) 10 mM Na₂HPO4, 1.8 mM KH₂PO₄ and 0.05% NaN₃, pH 7.2. PB(1000) 10mM Na₂HPO4, 1.8 mM KH₂PO₄, 0.05% NaN₃ and 1 M NaCl, pH 7.2. PBS 140 mMNaCl, 27 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ and 0.05% NaN₃. Thrombincleavage 60 mM NaCl, 60 mM KCl, 2.5 mM CaCl₂, 50 mM Tris, pH buffer 8.0.6.2 Cloning of Mini-IGFBP-5

Mini-IGFBP-5 (residues 40-92 of IGFBP-5) was subcloned from a vectorcontaining the complete sequence of IGFBP-5 into the BamHI and PstIrestriction sites of the pQE30-vector (Qiagen, Hilden, Germany).Restriction sites, a stop codon and 21 bases encoding an N-terminalthrombin cleavage site were introduced by means of PCR (Kalus, W., etal., EMBO J. 17 (1998) 6558-6572).

6.3 Mutagenesis of Mini-IGFBP-5

For introduction of mutations leading to substitution of LeU6, by Tyrand Leu₇₄ by Met, in vitro mutagenesis was performed using QuickChange™site-directed mutagenesis kit (Stratagene, La Jolla, Canada). Two setsof the following mutagenic oligonucleotide primers were designed foramplification of plasmid DNA and introduction of the desired pointmutations: FBP5LY: 5′-G GGG CTG CGC TGG TA C CCC CGG CAG (SEQ ID NO:1)GAC G-3′; RBP5LY: 5′-C GTC CTG CCG GGG G TA  GCA GCG CAG (SEQ ID NO:2)CCC C-3′; FBP5LM: 5′-CG CTG CAC GCC CTG A TG CAC GGC CGC (SEQ ID NO:3)GGG G-3′; RBP5LM: 5′-C CCC GCG GCC GTG CA T  CAG GGC GTG (SEQ ID NO:4)CAG CG-3′.

The changed codons (CTC into TAC in L₆₁Y mutant and CTG into ATG in L₇₄Mmutant) are presented in bold. Degenerated bases are underlined.

The reactions were set up according to the instructions found in themutagenesis kit manual. The PCR mixtures (50 μl) contained app 50 ng ofthe template (pQE30 (mini-IGFBP-5), prepared by means of mini prep spincolumns kit, Qiagen) and 125 ng of each of the two oligonucleotideprimers. Cycling parameters for the reactions were as follows: 30seconds at 95° C. followed by 13 cycles of 95° C. for 30 seconds, 55° C.for 1 minute and 68° C. for 7.5 min. The DpnI digestion and XL1-Bluesupercompetent cells transformation was carried out strictly accordingto the supplier's guidelines.

Two clones of each mutant were subjected to verification by automateddouble stranded sequencing, which proved the existence of the expectedsubstitutions in all 4 cases.

6.4 Expression of the Mutant Mini-IGFBP-5s

Electrocompetent cells BL21 were transformed with the construct carryingthe mutation. From a fresh plate, a 3-ml LB culture was started andgrown overday (6-7 h) in the presence of 300 μg ampicillin per ml at 37°C. From this culture 50 μl were used to inoculate 20 ml of MM. Thisculture was grown overnight (9-11 h). 1 l culture was inoculated in 1:50proportion. Expression of the protein was induced at OD₆₀₀≅0.8 byaddition of IPTG (1 mM final concentration). Cells were harvested after3 h (6000×G, 20 min at 4° C.).

6.5 Purification of Mini-IGFBP-5

Harvested cells were resuspended in buffer A (30 ml of the buffer wasused to resuspend cells from 11 culture) and incubated at roomtemperature with vigorous shaking (280 RMP) for 1 h to overnight. Thecells were opened by sonification (macrotip, 50% duty cycle, outputcontrol 70, 2×4 min). The cell extract was then centrifuged to pelletcell debris (65 000×G, 1 h at room temp.). The pH of the supernatant wasadjusted to the value of app. 8.0. The supernatant was then mixed withpre-equilibrated with buffer A Ni-NTA Superflow matrix (Qiagen),incubated with agitation for 1 h to overnight and then loaded onto anempty column (3 ml bed volume for 1 l culture). The column was washedwith buffer A followed by buffer B until a stable UV-absorption baseline. Bound proteins were fractionated with 100 ml pH gradient of bufferB and C. Collected fractions were analysed by tricine gelelectrophoresis (prior electrophoresis, the proteins were precipitatedwith 5% (w/v) TCA). Fractions containing mini-IGFBP-5 were pooled,concentrated on Amicon YM3 to 2-4 ml, and dialysed against 21 of bufferD overnight (100 μl excess of 2-mercaptoethanol was added to the sampleprior dialysis).

To promote refolding, the dialysed sample was diluted in 100 μl portionsinto freshly prepared, ice-cold buffer E, with vigorous stirring (inproportion 1 ml sample per 50 ml of buffer E), and left at 4° C. for 2-3days with stirring.

The sample was concentrated on Amicon YM3 to 15-25 ml, centrifuged toget rid of a precipitated material, and dialysed overnight into 41 ofbuffer PB containing 30 mM NaCl.

The solution was subsequently loaded onto pre-equilibrated with bufferPB (O) MonoS 5/5 HR cation-exchanger column (app. 1 ml) (AmershamPharmacia, Uppsala, Sweden) at a flow rate of 1 ml/min. The column waswashed with buffer PB (0). Proteins were eluted by 45 ml linear gradientof 0-70% NaCl, 1 ml fractions were collected.

The fractions containing mini-IGFBP-5 (as determined on the basis oftricine gel electrophoresis) were pooled, concentrated to 2-3 ml andloaded onto a pre-equilibrated with PBS Superdex 75 HiLoad 26/60 (app.320 ml) gel-filtration column (Pharmacia) at a flow rate of 0.6 ml/min.Mini-IGFBP-5 was eluted as a symmetrical, single pick. Fractionscontaining the protein were pooled and concentrated on centricon YM3.

6.6 Subcloning into pET-28a (+)

The reason for overall low expression of the proteins from the pQE30might be the fact that this vector is not well optimised for expressionin E. coli. For instance, the vector-encoded sequences contain a clusterof 3 rare codons just downstream from the initiator codon AUG (namely,AGA, GGA and TCG, encoding Arg, Gly and Ser, respectively). The numberof studies has indicated that excessive rare codon usage in a targetgene may be a cause for low level expression. The impact seems to bemost severe when multiple rare codons occur near the amino terminus andwhen they appear consecutively. Especially presence of the Arg codonsAGG and AGA can have severe effects on the level of protein production.The system seems to be also not well repressed (no extra copies of agene encoding Lac repressor), and the leaky expression may cause theobserved plasmid instability. The vector carries not very efficientselective marker, AmpR gene (bla), what makes possible rapidover-growing of a culture at a certain stage by cells lacking theunstable plasmid. The vector pET-28a (+) (Novagen) was then chosen as analternative for pQE30. The plasmid is well optimised for expression ofgenes in E. coli, carries a strong selective marker (KanR) and is stabledue to high level of repression of the target gene expression in theabsence of IPTG (in a non-DE3 lysogenic strain even in the presence ofthe inducer).

To subclone mini-IGFBP-5 wild type, L₆₁Y and L₇₄M from pQE30 to pET-28a,the relevant fragments were excised from the vector with BamHI andHindIII (HindIII cleavage site exists in pQE30 downstream from PstIsite). The excision was performed as double-digestion. Digested pETvector was 5′-dephosphorylated. Reaction mixtures were electrophorizedand bands corresponding to app. 200 bp fragments excised from pQE30(mini-IGFBP-5 wt, L₆₁,Y and L₇₄M) and app 5000 bp fragment of pET-28awere cut from 1% agarose gel and purified (gel extraction kit, Qiagen).The fragments were ligated (Ligation kit, Fermentas) and XL-1 BlueSupercompetent cells were transformed with the ligation mixture.

Restriction assay carried out subsequently on isolated plasmid DNArevealed presence of fragments of expected size (restriction enzymesNcoI and PstI were used, double digestion was performed. PstIrestriction site was introduced into the pET vector together with thefragment encoding mini-IGFBP-5).

Pilot-scale expression and purification experiment showed thatexpression of the protein of interest (mini-IGFBP-5 L₆₁Y in this case)is higher than the expression of the wild-type protein when pQE30 vectorwas used.

The proteins are expressed as double-fusions: they carry His-tagfollowed by T7-tag. It is possible to remove both tags by thrombincleavage. Mini-IGFBP-5 after cleavage by thrombin comprises thefollowing N-terminal amino acid sequence: GSALA (SEQ ID NO:7)(N-terminus of mini-IGFBP-5 starting from aa 40 with to additional aafrom cloning with thrombin cleavage site). Vector-derived amino acidsare underlined.

6.7 Subcloning of IGFBP4 from pKK177-3HB to pET-28a(+)

For subcloning of IGFBP4-2 into the NdeI and BamHI restriction sites ofthe pET-28a vector in-frame to a His-tag, following oligonucleotideswere designed for amplification of DNA by PCR: IBP4NdeI: 5′-CGG AGG AAAAA C   AT A TGG ATG AAG C- (SEQ ID NO:5) 3′ IBP4BamHI: 5′-GCC AAG CTT GGA  T C C AGG TCG AC-3′ (SEQ ID NO:6)

The restriction sites recognized by NdeI and BamHI are presented inbold. Degenerated bases are underlined.

The PCR mixture (50 μl) contained 124 ng of mixture of pKK177-3HB andPfdx500 repressor plasmid, 130 ng of each of the primers, 1 μl dNTP mixand 2.5 U Pfu Turbo DNA polymerase (Strategene). After initial step of30 sec. At 95° C., the reaction was cycled 30× at 95° C. for 30 seconds,55° C. for 1 min and 68° C. for 2 min. The product of PCR was purified(PCR purification kit, Qiagen), double-digested and electrophorised. Thebands corresponding to cleaved pET-28a and PCR product were excised fromthe gel and purified.

XL-1 Blue Supercompetent cells were transformed with the ligationmixture.

IGFBP4-2 is expressed as a N-terminal His-tag fusion protein. Afterthrombin cleavage, the protein comprises the following amino acidsequence: GSHMDEAIH . . . (SEQ ID NO:8). Vector derived amino acids areunderlined.

The same purification routine will be used for His-tagged IGFBP-4 as formini-IGFBP-5.

EXAMPLE 7

Identification of Chemical Non-Proteinaceous Compounds Binding toIGFBP-5 or IGF-I by Using the Coordinates of the Crystal Structure ofthe Complex of Both Molecules

FlexX version 1.9.0 was used to screen a substance library of ca. 90,000compounds in an ACD (Available Chemicals Directory; ACD-3D 2000),choosing compounds with a molecular weight of less than 550 Daltons andcontaining at least one of the atoms {N, O, F, or S}. Docking searcheswere conducted on both molecular surfaces of the IGFBP-5 interface. Topscoring hits as judged by the FlexX standard scoring function and theproximity to binding site protein atoms were selected and tested foractivity.

The top scoring compounds selected according to these these criteria forrelease of IGF-I from IGFBP-5 were:

-   Compound 1:    N1-(3,4-Dichlorophenyl)-2-[2-[5-(3,5-dichlorophenyl)-2H-1,2,3,-tetraazol-2YL]A    (MF: C16H11Cl4N7OS; MW: 491,1890 Da)-   Compound 2: F-MOC-Tyr(PO3H2)—OH(C₂₄H₂₂NO8P; MW: 483.4110)-   Compound 2A: Nα-FMOC-O-tert-butyl-L-tyrosine-   Compound 2B: Nα-FMOC-L-phenylalanine-   Compound 2C: Nα-FMOC-N-BOC-L-tryptophan-   Compound 2D: Nα-FMOC-L-leucine-   Compound 3:    4-(2,5-Dichlorophenylazo)-4′fluorosulfonyl-1-hydroxy-2-naphthanilide    (MF: C₂₃H₁₄Cl₂FN₃O₄S; MW: 518.3510)-   Compound 4B: 5-Amino-2[(4-amino-2-carboxyphenyl)thio]benzoic acid    (C₁₄H₁₂N₂O₄S; MW 304.3250)-   Compound 4C: 5-Amino-2[(2-carboxyphenyl)thio]benzoic acid    (C₁₄H₁₁NO4S; MW 289.3100)

EXAMPLE 8

Release of IGF-I from the Complex with IGFBP-5 by Selected CompoundsMeasured by IGF-I Binding to IGF-R Expressing Cells

Kalus, W., et al., in EMBO J. 17 (1998) 6558-6572, describe theinhibition of the binding of IGF-I to IGF-R expressing NIH 3T3 cells byformation of an inhibitory complex. This assay was used to investigatethe release of IGF-I from the inhibitory complex with IGFBP-5.

NIH 3T3 cells stably expressing human IGF-R were grown in culture dishesin Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calfserum. Cells were washed carefully with PBS and incubated with 5 ml of50 mM EDTA in PBS for 45 min. Cells were removed from the plate, washedonce with PBS and once with binding buffer (100 mM HEPES pH 7.6, 120 mMNaCl, 5 mM KCl, 1.2 mM MgSO 4, 1 mM EDTA, 10 mM glucose, 15 mM sodiumacetate, 1% dialysed BSA), and resuspended in binding buffer todetermine the cell number. 5 pM ¹²⁵ I-IGF-I (Amersham) was preincubatedwith either 10 or 100 nM IGFBP-5 alone or in combination with 33 μM ofthe different compounds 1,2,3,4B and 4C at 4° C. for 1 h. Then 400 μl ofthe cell suspension corresponding to 2×10⁵ cells were added to give atotal volume of 500 μl. After 12 h incubation at 4° C., cells werewashed with binding buffer (at 4° C.). Free hormone was removed byrepeated centrifugation and resuspension in the binding buffer. The 125I radioactivity bound to the cells was determined in a gamma-counter.

As shown in FIG. 7 the labeled IGF-I binds to NIH 3T3 cells in theabsence of IGFBP-5 and cell binding is inhibited by the addition ofIGFBP-5. Preincubation of the complex of IGFBP-5 and IGF-I with theselected compounds results in release of IGF-I from the complex bycompound 3 and consequently binding of IGF-I to the IGF-R expressingcells.

EXAMPLE 9

Release of IGF-I from the Complex with IGFBP-5 by Selected CompoundsMeasured by IGF-R Activation

Kalus, W., et al., in EMBO J. 17 (1998) 6558-6572, describe theinhibition of the activation and autophosphorylation of the IGF-R byIGF-I in the presence of IGFBP-5. This assay was used to furtherinvestigate the release of IGF-I from the inhibitory complex withIGFBP-5 by compound 3. Binding of compound 3 to IGFBP-5 and dissociationof the complex of the binding protein with IGF-I should result in anactivation and autophosphorylation of the IGF-R in the presence ofIGFBP-5.

Confluent monolayers of the NIH 3T3 cells stably expressing human IGF-Rin 3.5 cm dishes were starved in DMEM containing 0.5% dialysed fetalcalf serum. After 48 h, cells were incubated without any hormone or with10 nM IGF-I. Samples were preincubated with 100 nM IGFBP-5 andincreasing concentrations of compound 3 from 0 to 50 μM at roomtemperature for 1 h. After a 10 min stimulation at 37° C., the mediumwas removed and cells were lysed with 250 μl of lysing buffer (20 mMHEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1% NP-40, 1.5 mM MgCl 2, 1 mMEGTA), 10 mM sodium orthovanadate, and protease inhibitor cocktailComplete (Roche Molecular Biochemicals) for 10 min on ice. Subsequently,cells were scraped off the plate and the insoluble material wasseparated by centrifugation for 20 min at 4° C. The proteinconcentration of the supernatant was determined using the BCA kit fromPierce according to the manufacturer's instructions. Equal proteinconcentration was incubated with the SDS sample buffer (63 mM Tris-HClpH 6.8, 3% SDS, 10% glycerol, 0.05% bromophenolblue, 100 mM DTT), boiledfor 5 min and loaded on a 7.5% SDS-polyacrylamide gel. Afterelectrophoresis the proteins were transferred on a nitrocellulosemembrane which first was blocked for 1 h with the 3% BSA containingphosphate-buffered saline-Tween (PBST), then incubated overnight with 1mg/ml monoclonal anti-phosphotyrosine antibody 4G10 (UpstateBiotechnology), polyclonal anti-phospho-AKT antibody (New EnglandBiolabs) or polyclonal anti-IGF-R(C-20, Santa Cruz Biotechnology) inPBST that contained 3% BSA. Unbound antibody was removed by extensivewashing. The blot was then incubated with 1:10 000 diluted anti-mouseIgG-specific antibody or 1:5000 diluted anti-rabbit specific antibodyconjugated with horse radish peroxidase (both Roche MolecularBiochemicals). The immunoblot was developed using the ECL kit fromAmersham.

As shown in FIG. 8 the autophosphorylation of IGF-R by IGF-I isinhibited in the presence of IGFBP-5. The addition of compound 3 to theinactive complex of IGFBP-5 and IGF-I results in an increasedautophosphorylation of the receptor at 50 uM compound 3.

EXAMPLE 10

Detection of Ligand Binding

Ligand binding was detected by acquiring ¹⁵N—HSQC spectra. All NMRspectra were acquired at 300 K on Bruker DRX600 spectrometer. Thesamples for NMR spectroscopy were concentrated and dialyzed against PBSbuffer. Typically, the sample concentration was varied from 0.3 to 1.0mM. Before measuring, the sample was centrifuged in order to sedimentaggregates and other macroscopic particles. 450 μl of the proteinsolution were mixed with 50 μl of D₂O (5-10%) and transferred to an NMRsample tube. The stock solutions of compounds were 100 mM either inwater or in perdeuterated DMSO. pH was maintained constant during thewhole titration. The binding was monitored by observation of the changesin the ¹⁵N—HSQC spectrum. Dissociation constants were obtained bymonitoring the chemical shift changes of the backbone amide of severalamino acid residues (Table 9) as a function of ligand concentration.Data were fit using a single binding site model. In the same waydissociation constants for derivatives of compound 2 are estimated(Table 10). TABLE 9 Dissociation constant calculations for compound 2 orDMSO binding to IGFBP-5 using data from distinct amino acid residuesligand in DMSO K_(D) ligand in PBS K_(D) residue [mM] [mM] DMSO K_(D)[mM] Y50 1.58 ± 0.09 1.82 ± 0.95 648 ± 370 L73 1.31 ± 0.17 2.93 ± 1.41541 ± 306 S85 1.38 ± 0.10 2.33 ± 0.94 650 ± 373 Y86 1.90 ± 0.17 1.72 ±0.99 783 ± 498 R87 1.64 ± 0.12 2.36 ± 1.00 921 ± 662 K91 2.42 ± 0.182.12 ± 1.03 719 ± 434 average: 1.71 ± 0.37 2.21 ± 0.40 710 ± 120

TABLE 10 Dissociation constants calculated for compound 2 and itsderivatives binding to IGFBP-5 using changes in chemical shift for theresidue L81 compound chemical name K_(D) [mM] 2Nα-FMOC-O-phospho-L-tyrosine  2.78 ± 0.30 2ANα-FMOC-O-tert-butyl-L-tyrosine  0.718 ± 0.079 2BNα-FMOC-L-phenylalanine  1.075 ± 0.507 2C Nα-FMOC-N-BOC-L-tryptophan0.0432 ± 0.0115 2D Nα-FMOC-L-leucine  1.088 ± 0.519

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1. A crystal suitable for X-ray diffraction, comprising a complex ofinsulin-like growth factor I or II (IGF) and a polypeptide consisting ofthe amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2, theamino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, theamino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or afragment thereof consisting at least of the 9^(th) to 12^(th) cysteineof IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the7^(th) to 10^(th) cysteine of IGFBP-6, to form a complex which exhibitsrestricted conformation mobility.
 2. A crystal of claim 1, whicheffectively diffracts X-ray for the determination of the atomiccoordinates of the complex to a resolution of 1.5 to 3.5 Å. 3.(Canceled)
 4. A method for producing a crystal suitable for X-raydiffraction, comprising (a) contacting IGF with a polypeptide consistingof the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2,the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, theamino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or afragment thereof consisting at least of the 9^(th) to 12^(th) cysteineof IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the7^(th) to 10^(th) cysteine of IGFBP-6, to form a complex which exhibitsrestricted conformation mobility, and (b) obtaining a crystal from thecomplex so formed suitable for X-ray diffraction.
 5. A method for thedetermination of the atomic coordinates of a crystal suitable for X-raydiffraction obtained by (a) contacting IGF with a polypeptide consistingof the amino acids 39-91 of IGFBP-1, the amino acids 55-107 of IGFBP-2,the amino acids 47-99 of IGFBP-3, the amino acids 39-91 of IGFBP-4, theamino acids 40-92 of IGFBP-5, or the amino acids 40-92 of IGFBP-6 or afragment thereof consisting at least of the 9^(th) to 12^(th) cysteineof IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the7^(th) to 10^(th) cysteine of IGFBP-6, to form a complex which exhibitsrestricted conformation mobility; and (b) obtaining a crystal from thecomplex so formed suitable for X-ray diffraction; (c) determining theatomic coordinates of said crystal.
 6. A method for identifying a mutantof IGFBP (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 or IGFBP-6 or amutant of a fragment thereof consisting at least of the 9^(th) to12^(th) cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or atleast of the 7^(th) to 10^(th) cysteine of IGFBP-6) having an enhancedbinding affinity for IGF, comprising (a) constructing athree-dimensional structure of the complex of IGF and a polypeptideconsisting of the amino acids 39-91 of IGFBP-1, the amino acids 55-107of IGFBP-2, the amino acids 47-99 of IGFBP-3, the amino acids 39-91 ofIGFBP4, the amino acids 40-92 of IGFBP-5, or the amino acids 40-92 ofIGFBP-6 consisting at least of the 9^(th) to 12^(th) cysteine ofIGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 or at least of the 7^(th)to 10^(th) cysteine of IGFBP-6, based on the atomic coordinates of acrystal consisting of IGFI and said IGFBP or a fragment thereof; (b)employing said three-dimensional structure and modeling methods toidentify said mutant of an IGFBP in which a residue within a distance of5 Å to a hydrophobic amino acid residue of IGF is modified in that thehydrophobic interaction between IGF and said mutant of IGFBP isenhanced; (c) producing said mutant; (d) assaying said mutant todetermine said enhanced binding affinity for IGF.
 7. A method foridentifying a mutant of IGFBP-5 with enhanced binding affinity forIGF-1, said method comprising (a) constructing a three-dimensionalstructure of the complex of IGF and IGFBP-5 defined by the atomiccoordinates shown in FIGS. 5 and 6; (b) employing said three-dimensionalstructure and modeling methods to identify an amino acid residue inIGFBP-5 within a distance of 5 Å or shorter to an amino acid residue ofIGFI, wherein said residue of IGFBP-5 can be modified hydrophobically inthat the hydrophobic interaction between IGF and IGFBP-5 is enhanced;(c) producing said mutant; (d) assaying said mutant to determine saidenhanced binding affinity for IGF.
 8. A mutant of IGFBP containing oneor more of the mutations as depicted in Tables 1 to
 6. 9. A mutant ofIGFBP containing one or more mutations of amino acid residues 49, 70and/or 73 corresponding to IGFBP-5 sequence alignment according toTables 1 to
 6. 10. A method for identifying a non-proteinaceous compoundcapable of binding to IGFBP, comprising (a) constructing athree-dimensional structure of a complex of insulin-like growth factor 1or 11 and a polypeptide consisting of the amino acids 40-92 ofinsulin-like growth factor binding protein 5, amino acids 39-91 ofIGFBP-1, amino acids 55-107 of IGFBP-2, amino acids 47-99 of IGFBP-3,amino acids 39-91 of IGFBP-4, amino acids 40-92 of IGFBP-5, amino acids40-92 of IGFBP-6 or a fragment thereof consisting at least of the 9^(th)to 12^(th) cysteine of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, or IGFBP-5 orat least of the 7^(th) to 10^(th) cysteine of IGFBP-6, based on theatomic coordinates of a crystal consisting of IGF-I and said IGFBP; (b)employing said three-dimensional structure and modeling methods toidentify a non-proteinaceous compound forming a complex with said IGFBPby hydrophobic binding with amino acids 49, 50, 70, 71 and 74 in thecase of IGFBP-5 and in the case of IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4and IGFBP-6 with the corresponding amino acids according to Table 7; (c)producing said compound; (d) determining the binding between thecompound and said IGFBP.
 11. A crystal of claim 1, wherein the crystalis arranged in the cubic space group P2,3 having unit cell dimensions of74.385 Å.×74.385 Å×74.385 Å.
 12. A crystal of claim 2, wherein thecrystal is arranged in the cubic space group P2,3 having unit celldimensions of 74.385 Å.×74.385 Å×74.385 Å.